Activation of the phosphoinositide-3 kinase (PI-3K) pathway constitutes one of the most important mechanisms that regulates important cellular functions such as gene expression, cell cycle progression, cell growth, and differentiation (Dygas and Baranska, Acta Biochim. Pol. 48:541-549 (2001)). Modification of AKT and other PI-3K downstream kinases in the cytosol is mediated by 3-phosphoinositide dependent kinase 1 (PDK1), a serine/threonine kinase originally identified as a kinase critical for AKT activation loop phosphorylation and activation (Cohen et al., FEBS Lett., 410:3-10 (1997)).
Substrates of PDK1 include many of the AGC family of protein kinases (the cAMP-dependent, cGMP-dependent, and protein kinase C), including AKT/PKB, p70S6K, cyclic AMP-dependent protein kinase, protein kinase C, serum and glucocorticoid-inducible kinase (SGK), p90 ribosomal protein kinase (RSK), p21-activated kinase-1 (PAK1) PRK1/2, and others (Wick and Liu, Curr. Drug Targets Immune Endocr. Metabol. Disord. 1:209-221 (2001); Mora et al., Semin. Cell Dev. Biol. 15:161-170 (2004)). However, recent in vivo studies with PDK1(−/−) and PDK1(−/+) mice showed that the most physiologically relevant substrates of PDK1 are AKT, p70S6K and RSK (Lawlor et al EMBO J. 21:3728-3738 (2002); Williams et al., Curr. Biol. 10:439-448 (2000)). Activation of these critical PDK1 substrates leads to an increase in glucose uptake, protein synthesis, and inhibition of pro-apoptotic proteins.
Regulation of AKT is a best studied example of the PI3K-dependent activity of PDK1. Specific inhibitors of PI3K or dominant negative AKT mutants abolish survival-promoting activities of growth factors or cytokines. It has been previously described that inhibitors of PI3K (LY294002 or wortmannin) blocked the activation of AKT by upstream kinases. In addition, introduction of constitutively active PI3K or AKT mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al., Mol. Cell Biol. 17(3):1595-1606 (1997); Dudek et al, Science 275(5300):661-665 (1997)).
Analysis of AKT levels in human tumors revealed that AKT is overexpressed in a significant number of ovarian (Cheung et al., Proc. Natl. Acad. Sci. U.S.A. 89:9267-9271 (1992)) and pancreatic cancers (Cheung et al., Proc. Natl. Acad. Sci. U.S.A. 93:3636-3641 (1996)). Also, AKT was found to be overexpressed in breast and prostate cancer cell lines (Nakatani et al. J. Biol. Chem. 274:21528-21532 (1999)).
A significant number of cancers possess mutations in genes that result in elevation of cellular levels of phosphatidylinositol 3,4,5-triphosphate (PIP3), a product of PI3K. One of the most common mutations giving rise to higher levels of PIP3 is in the PIP3 3-phosphatase PTEN gene (Leslie and Downes, Cell Signal. 14:285-295 (2002); Cantley, Science 296:1655-1657 (2002)). Increased levels of PIP3 result in over-activation of AKT and p70S6K kinases, which are thought to function as major driving forces in promoting the uncontrolled proliferation and enhanced survival of these cells. Germline mutations of PTEN are responsible for human cancer syndromes such as Cowden disease (Liaw et al., Nature Genetics 16:64-67 (1997)). PTEN is deleted in a large percentage of human tumors and tumor cell lines without functional PTEN show elevated levels of activated AKT (Li et al. supra, Guldberg et al., Cancer Research 57:3660-3663 (1997), Risinger et al., Cancer Research 57:4736-4738 (1997)).
Three members of the AKT/PKB subfamily of second-messenger regulated serine/threonine protein kinases have been identified and termed AKT1, AKT2 and AKT3. The isoforms are homologous, particularly in regions encoding the catalytic domains. AKTs are activated by phosphorylation events occurring in response to PI3K signaling. PI3K phosphorylates membrane inositol phospholipids, generating the second messengers phosphatidylinositol 3,4,5-triphosphate (PIP3) and phosphatidylinositol 3,4-diphosphate, which have been shown to bind to the PH domain of AKT. The current model of AKT activation proposes recruitment of the enzyme to the membrane by PIP3. PDK1 also possesses the PH domain and it is postulated that co-localization of AKT and PDK1 at the membrane allows for AKT modification and activation by PDK1 and possibly other kinases (Hemmings, Science 275:628-630 (1997); Hemmings, Science 276:534 (1997); Downward, Science 279:673-674 (1998)).
Phosphorylation of AKT1 occurs on two regulatory sites, Thr308 by PDK1 in the catalytic domain activation loop and Ser473 (most probably by TORC2 mTOR complex) near the carboxy terminus (Alessi et al., EMBO J. 15:6541-6551 (1996); Meier et al., J. Biol. Chem. 272:30491-30497 (1997)).
Important non-PI3K-dependent physiological substrates of PDK1, p90 ribosomal protein S6 kinases (RSKs) have been recently implicated in promoting tyrosine receptor-induced hematopoetic transformation (Kang et al., Cancer Cell 12:201-214 (2007)). PDK1 activates RSK by phosphorylating its amino terminal kinase domain in an ERK-dependent manner (Cohen et al., Nature Chem. Biol. 3(3):156-160 (2007)).
Also, recent studies revealed additional roles of PDK1 that could be important during tumorigenesis and metastasis, such as cell motility and migration (Primo et al., J. Cell Biol. 176(7):1035-1047 (2007); Pinner and Sahai, Nature Cell Biol. 10(2):127-137 (2008)).
Taken together, these observations suggest that an inhibitor of PDK1 might be beneficial for treatment of cancer cells possessing (but not limited to) constitutively activated AGC kinases.